Mathematical morphologic analysis of liver cirrhosis. Correlation with etiology, clinical score and hepatocellular carcinoma.

Remodeling of the cirrhotic liver was studied retrospectively by mathematical morphologic methods in 75 autopsy cases (40 alcoholic, 17 hepatitis B virus (HBV)-related and 18 cryptogenetic cirrhosis), including 28 hepatocellular carcinomas. The aim was to obtain objective measurements of cirrhotic patterns that could be correlated with liver function evaluated by the Pugh-Child score, establish the relationship among different morphogenetic features and evaluate the implications of an objective classification of cases by numerical taxonomy in terms of their etiology, liver function and malignant transformation. The results indicate that the Pugh-Child score was closely related to the global amount of fibrosis or to the percentage of regenerative nodules < 0.8 mm in diameter. In contrast, the higher the percentage of lobular-sized regenerative nodules (0.8-1.6 mm), the better the functional score, suggesting that they are probably residual lobules, albeit completely surrounded by fibrous tissue, rather than true regenerative pseudolobules. The four groups of cases obtained by numerical taxonomy (cluster analysis) showed different distributions for alcoholic and HBV-related cirrhosis. The pattern of the latter was practically analogous to that in classically labeled cryptogenetic cirrhosis, suggesting its viral etiology. Taxonomic classification had functional implications. The Pugh-Child score showed a definite relationship with the different clusters obtained. The incidence of malignant transformation gradually decreased from group G1 to G4, with a steeper descent between G2 and G3. These results might contribute to a more dynamic concept of morphologic changes in liver biopsies from patients with cirrhosis.     

Decreased Plasma Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Levels are Linked with Disease Severity in Patients with Liver Cirrhosis

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide that has been found to reduce liver inflammation and protect liver from steatosis. We aimed to measure the level of plasma PACAP in patients with liver cirrhosis and to correlate its level with disease severity and laboratory parameters. 113 patients with clinically diagnosed liver cirrhosis and 110 healthy individuals were selected. Child–Pugh score was performed to evaluate disease severity. Liver biopsy was performed using the Knodell modified histology activity index (KmHAI). The L3 skeletal muscle index (L3SMI) was calculated to evaluate the nutrition status. Plasma PACAP concentrations were detected using ELISA. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the sensitivity and specificity of PACAP for detecting the severity of liver cirrhosis. Plasma PACAP levels were found to be significantly downregulated in patients with liver cirrhosis compared with healthy controls. The case group included 38 Child–Pugh A patients, 39 Child–Pugh B patients, and 36 Child–Pugh C patients. Cirrhosis patients with Child–Pugh C had significantly lower plasma PACAP levels compared with those with Child–Pugh B and A. Cirrhosis patients with Child–Pugh B demonstrated markedly decreased plasma PACAP concentrations compared with those with Child–Pugh A. ROC curve analysis indicated that low level of plasma PACAP may act as a potential indicator for disease progression of liver cirrhosis determined by Child–Pugh classification at both compensation stage (Child–Pugh A developed to Child–Pugh B) and decompensation stage (Child–Pugh B developed to Child–Pugh C).In addition, plasma PACAP levels were inversely associated with KmHAI and ALT as well as AST levels. In both male and female patients, plasma PACAP levels were positively related to L3SMI score. Decreased plasma PACAP levels are linked with disease severity in patients with liver cirrhosis. Reduced plasma PACAP may serve as a biomarker for detection and assessment of the severity of liver cirrhosis.

     

Effects of thermal acclimation on transcriptional responses to acute heat stress in the eurythermal fish Gillichthys mirabilis (Cooper)

The capacities of eurythermal ectotherms to withstand wide ranges of temperature are based, in part, on abilities to modulate gene expression as body temperature changes, notably genes encoding proteins of the cellular stress response. Here, using a complementary DNA microarray, we investigated the sequence in which cellular stress response-linked genes are expressed during acute heat stress, to elucidate how severity of stress affects the categories of genes changing expression. We also studied how prior acclimation history affected gene expression in response to acute heat stress. Eurythermal goby fish (Gillichthys mirabilis) were acclimated to 9 ± 0.5, 19 ± 0.5, and 28 ± 0.5°C for 1 mo. Then fish were given an acute heat ramp (4°C/h), and gill tissues were sampled every +4°C to monitor gene expression. The average onset temperature for a significant change in expression during acute stress increased by ～2°C for each ～10°C increase in acclimation temperature. For some genes, warm acclimation appeared to obviate the need for expression change until the most extreme temperatures were reached. Sequential expression of different categories of genes reflected severity of stress. Regardless of acclimation temperature, the gene encoding heat shock protein 70 (HSP70) was upregulated strongly during mild stress; the gene encoding the proteolytic protein ubiquitin (UBIQ) was upregulated at slightly higher temperatures; and a gene encoding a protein involved in cell cycle arrest and apoptosis, cyclin-dependent kinase inhibitor 1B (CDKN1B), was upregulated only under extreme stress. The tiered, stress level-related expression patterns and the effects of acclimation on induction temperature yield new insights into the fundamental mechanisms of eurythermy.     

Multigene Tracking of Hepatitis C Virus Quasispecies after Liver Transplantation: Correlation of Genetic Diversification in the Envelope Region with Asymptomatic or Mild Disease Patterns

To investigate the role of hepatitis C virus (HCV) quasispecies mutation in the pathogenesis of HCV infection, we analyzed changes in the genetic diversity of HCV genomes in 22 patients before and after liver transplantation by using heteroduplex mobility assay (HMA) technology. All patients were infected with HCV genotype 1 and developed high-titer posttransplant viremia. Each patient was classified according to the severity of posttransplant hepatitis, as assessed by standard biochemical and histological criteria. HCV quasispecies were characterized by HMA analysis of eight separate subgenomic regions of HCV, which collectively comprise 44% of the entire genome. The glycoprotein genes E1 and E2, as well as the nonstructural protein genes NS2 and NS3, had the greatest genetic divergence after liver transplantation (the change in the heteroduplex mobility ratio [HMR] ranged from 2.5 to 7.0%). In contrast, genes encoding the core, NS4, and NS5b proteins had the least amount of genetic divergence after liver transplantation (range, 0.3 to 1.2%). The E1/E2 region showed the greatest change in genetic diversity after liver transplantation, and the change in HMRs was 2.5- to 3.3-fold greater in patients with asymptomatic or moderate disease than in those with severe disease. The E1-5′ region of HCV quasispecies isolated from patients in the asymptomatic group had a significantly greater degree of diversification after liver transplantation than the same regions of HCV quasispecies isolated from patients in the severe disease group (P = 0.05). While changes in the genetic diversity of some nonstructural genes were also greater in asymptomatic patients or in patients with mild disease than in patients with severe disease, the results were not significant. Data from this cohort demonstrate that greater rates of HCV quasispecies diversification are associated with mild or moderate liver disease activity in this immunosuppressed population.

Hepatitis C virus (HCV), a member of the Flaviviridae family, is known to be a major causative agent of chronic liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (27). Chronic hepatitis C is now recognized as the leading indication for orthotopic liver transplantation in the United States, with nearly 100% of HCV-infected liver transplant recipients developing recurrent viremia after transplantation (4, 17, 22, 32, 44).The HCV genome is a single-stranded, positive-sense RNA of about 9.5 kb, which exists as a viral quasispecies in infected humans (6, 25, 31, 41). HCV quasispecies are characterized by extensive genetic mutation in the hypervariable region 1 (HVR1) of the second envelope glycoprotein gene (E2) (25, 41). Mutation of this region of the genome is believed to be associated with viral persistence via immune escape mechanisms (15, 16, 29, 42). The role of evolution of HCV quasispecies in the development of chronic hepatitis C is currently unknown.HCV-infected liver transplant recipients offer an opportunity to study the evolution of HCV quasispecies in a new host tissue and to assess the role of quasispecies diversification in the development of posttransplant hepatitis. In a previous study (23) of HCV-infected liver transplant recipients, we found that in the three patients who developed severe, recurrent hepatitis, quasispecies major variants present in pretransplant serum samples were efficiently propagated after liver transplantation and during acute and chronic posttransplant hepatitis. In contrast, in the two asymptomatic cases, we observed rapid depletion of pretransplant quasispecies major variants from posttransplant serum samples, followed by the emergence of quasispecies minor variants. These data suggested that the evolution of HCV quasispecies after liver transplantation may be related to posttransplant disease severity.In order to extend our previous findings and to address the hypothesis that mutation of other HCV genes also correlates with severity of posttransplant hepatitis C, we analyzed the pre- and posttransplant HCV quasispecies in 22 HCV-infected liver transplant recipients. The 22 patients were selected based on two virological criteria: all patients were infected with HCV genotype 1 before and after liver transplantation, and all patients developed recurrent, high-titer HCV viremia within 30 days posttransplant. Additionally, at least five posttransplant liver biopsies were available in each case to evaluate the histopathologic course of posttransplant hepatitis C. Pre- and posttransplant HCV quasispecies were characterized by using the heteroduplex mobility assay (HMA) to analyze eight different regions of the viral genome.     

Serum Sphingolipid Variations Associate with Hepatic Decompensation and Survival in Patients with Cirrhosis

Background

Sphingolipids constitute bioactive molecules with functional implications in liver homeostasis. Particularly, ablation of very long chain ceramides in a knockout mouse model has been shown to cause a severe hepatopathy.

Methods

We aimed to evaluate the serum sphingolipid profile of 244 patients with cirrhosis prospectively followed for a median period of 228±217 days via mass spectrometry.

Results

We thereby observed a significant decrease of long and very long chain ceramides, particularly of C24ceramide, in patients with increasing severity of cirrhosis (p<0.001). Additionally, hydropic decompensation, defined by clinical presentation of ascites formation, was significantly correlated to low C24ceramide levels (p<0.001) while a significant association to hepatic decompensation and poor overall survival was observed for low serum concentrations of C24ceramide (p<0.001) as well. Multivariate analysis further identified low serum C24ceramide to be independently associated to overall survival (standard beta = -0.001, p = 0.022).

Conclusions

In our current analysis serum levels of very long chain ceramides show a significant reciprocal correlation to disease severity and hepatic decompensation and are independently associated with overall survival in patients with cirrhosis. Serum sphingolipid metabolites and particularly C24ceramide may constitute novel molecular targets of disease severity, hepatic decompensation and overall prognosis in cirrhosis and should be further evaluated in basic research studies.     

Expression of apolipoprotein AI mRNA in peripheral white blood cells of patients with alcoholic liver disease

Because (i) changes in plasma and liver mRNA of apolipoprotein (apo) AI have been observed in patients with alcoholic liver disease, (ii) apo AI mRNA can be induced in non-hepatic tissues, and (iii) apolipoproteins expression is influenced by plasma colloid osmotic pressure (P(CO)) and viscosity (eta), we analyzed the Apo AI mRNA expression in the peripheral white blood cells (PWBC), P(CO), and eta in control volunteers (C), patients with liver cirrhosis (LC), and cirrhotic patients with superimposed alcoholic hepatitis (LC+AH). We found that apo AI mRNA is expressed in the PWBC in 20% of C and it is induced 1.5 fold in 66.6% of LC and 1.95 fold in 85% of LC+AH. A significant decrease of P(CO) in LC and LC + AH (14.8 +/- 2.4 and 16.2 +/- 2.4 mm Hg, respectively) compared to C (27.9 +/- 2 mm Hg) was observed. By contrast, eta was mildly increased from 1.7389 +/- 0.07 in C to 1.8022 +/- 0.154 in LC and 1.9030 +/- 0.177 in LC+AH. No significant correlation was found between P(CO) and eta with apo AI mRNA but with lipid profile. In conclusion, apo AI mRNA expression in PWBC is associated to liver disease severity and could be an indirect indicator of alcoholic liver damage.     

The locus coeruleus is directly implicated in L-DOPA-induced dyskinesia in parkinsonian rats: an electrophysiological and behavioural study

Despite being the most effective treatment for Parkinson's disease, L-DOPA causes a development of dyskinetic movements in the majority of treated patients. L-DOPA-induced dyskinesia is attributed to a dysregulated dopamine transmission within the basal ganglia, but serotonergic and noradrenergic systems are believed to play an important modulatory role. In this study, we have addressed the role of the locus coeruleus nucleus (LC) in a rat model of L-DOPA-induced dyskinesia. Single-unit extracellular recordings in vivo and behavioural and immunohistochemical approaches were applied in rats rendered dyskinetic by the destruction of the nigrostriatal dopamine neurons followed by chronic treatment with L-DOPA. The results showed that L-DOPA treatment reversed the change induced by 6-hydroxydopamine lesions on LC neuronal activity. The severity of the abnormal involuntary movements induced by L-DOPA correlated with the basal firing parameters of LC neuronal activity. Systemic administration of the LC-selective noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine did not modify axial, limb, and orolingual dyskinesia, whereas chemical destruction of the LC with ibotenic acid significantly increased the abnormal involuntary movement scores. These results are the first to demonstrate altered LC neuronal activity in 6-OHDA lesioned rats treated with L-DOPA, and indicate that an intact noradrenergic system may limit the severity of this movement disorder.     

Rat liver NAD(P)H: quinone reductase nucleotide sequence analysis of a quinone reductase cDNA clone and prediction of the amino acid sequence of the corresponding protein

We have determined the nucleotide sequence of a cDNA clone, pDTD55, complementary to rat liver quinone reductase mRNA (Williams, J.B., Lu, A.Y.H., Cameron, R.G., and Pickett, C.B. (1986) J. Biol. Chem. 261, 5524-5528). The cDNA clone contains an open reading frame of 759 nucleotides encoding a polypeptide comprised of 253 amino acids with a Mr = 28,564. To verify the predicted amino acid sequence of quinone reductase, we have been able to align the amino acid sequences of a cyanogen bromide digest of the purified enzyme to the sequence deduced from the cDNA clone. A comparison of the quinone reductase sequence with other known flavoenzymes did not reveal a significant degree of amino acid sequence homology. These data suggest that the quinone reductase gene has evolved independently from genes encoding other flavoenzymes.     

Integrating the contributions of mitochondrial oxidative metabolism to lipotoxicity and inflammation in NAFLD pathogenesis

The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing globally. NAFLD includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). NASH is the pathological form of the disease characterized by liver steatosis, inflammation, cell injury, and fibrosis. A fundamental contributor to NASH is the imbalance between lipid accretion and disposal. The accumulation of liver lipids precipitates lipotoxicity and the inflammatory contributions to disease progression. This review defines the role of dysregulated of lipid disposal in NAFLD pathophysiology. The characteristic changes in mitochondrial oxidative metabolism pathways and the factors promoting these changes across the spectrum of NAFLD severity are detailed. This includes pathway-specific and integrative perturbations in mitochondrial β-oxidation, citric acid cycle flux, oxidative phosphorylation, and ketogenesis. Moreover, well-recognized and emerging mechanisms through which dysregulated mitochondrial oxidative metabolism mediates inflammation, fibrosis, and disease progression are highlighted.     

Effects of colchicine on liver functions of cirrhotic rats: beneficial effects result from stellate cell inactivation and inhibition of TGF beta1 expression

Liver cirrhosis (LC) is a chronic disease with high mortality rate and its pathophysiology includes hepatic parenchymal cell destruction, connective tissue formation, and nodular regeneration. Colchicine has been used in liver diseases as an anti-inflammatory and anti-fibrotic drug. However, there is controversy over the beneficial effects of colchicine in LC treatment. In the present study, we injected rats with multiple doses of dimethylnitrosamine for 4 weeks and used rats with severe LC to determine whether colchicine treatment improved liver functions and resolved cirrhotic nodules. Colchicine (30-150microg/kg per day, i.p., for 4 weeks) failed to significantly increase the survival rate of LC rats. Animals were subjected to blood biochemical, liver histopathological and immunochemical analyses. The plasma albumin level, decreased in cirrhotic rats, was restored by colchicine treatment along with reduction of ascites. Colchicine decreased the accumulated extracellular matrix and the multiple fibrotic nodules formed in cirrhotic liver, and eliminated alpha-smooth muscle actin (alpha-SMA)-positive cells. In activated stellate cells, colchicine inhibited alpha-SMA and transforming growth factor-beta1 (TGFbeta1) expression. The results of the present study showed that colchicine resolves cirrhotic nodules and accumulated fibers in the liver of LC rats, but failed to significantly improve the survival rate of LC animals, and that the beneficial effects of colchicine in cirrhotic animals result from stellate cell inactivation and inhibition of TGFbeta1 expression.     

Increased hepatic synthesis and dysregulation of cholesterol metabolism is associated with the severity of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is associated with increased cardiovascular and liver-related mortality. NAFLD is characterized by both triglyceride and free cholesterol (FC) accumulation without a corresponding increment in cholesterol esters. The aim of this study was to evaluate the expression of cholesterol metabolic genes in NAFLD and relate these to disease phenotype. NAFLD was associated with increased SREBP-2 maturation, HMG CoA reductase (HMGCR) expression and decreased phosphorylation of HMGCR. Cholesterol synthesis was increased as measured by the circulating desmosterol:cholesterol ratio. miR-34a, a microRNA increased in NAFLD, inhibited sirtuin-1 with downstream dephosphorylation of AMP kinase and HMGCR. Cholesterol ester hydrolase was increased while ACAT-2 remained unchanged. LDL receptor expression was significantly decreased and similar in NAFLD subjects on or off statins. HMGCR expression was correlated with FC, histologic severity of NAFLD and LDL-cholesterol. These data demonstrate dysregulated cholesterol metabolism in NAFLD which may contribute to disease severity and cardiovascular risks.     

高尔基体跨膜糖蛋白73高表达诱发脂肪肝的脂类特性分析

脂肪性肝病是最常见的慢性肝脏疾病,脂质代谢异常是脂肪性肝病发生的重要原因。为研究高尔基体糖蛋白(Golgi protein 73, GP73)对肝脏脂质代谢的影响,选用八周龄C57BL/6J小鼠通过尾静脉注射搭载GP73的腺相关病毒(AAV-GP73),构建肝脏特异性高表达GP73的小鼠,通过对肝脏进行脂质代谢组学分析发现小鼠肝脏中的脂质尤其是甘油三酯明显增加。京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes, KEGG)富集分析显示,GP73通过引起脂代谢产物的改变导致诸多与细胞代谢活动相关的信号通路出现紊乱,特别是与人类密切相关的疾病如Ⅱ型糖尿病、非酒精性脂肪性肝病(NAFLD)和癌细胞胆碱代谢更可能发生失调。研究表明,GP73可能通过参与调控脂类代谢并促进肝脏内脂质积累诱发脂肪肝。     

Immune-relevant genes expressed in rainbow trout following immunisation with a live vaccine against Ichthyophthirius multifiliis

Rainbow trout Oncorhynchus mykiss were immunised by intra-peritoneal injection using a live vaccine based on Ichthyophthirius multifiliis (Ich) theronts, which previously has shown protection against white spot disease. Samples were taken pre-vaccination and on Day 1, 7, 21 and 28 post-immunisation (p.i.). Expression of immune relevant genes in the liver, spleen and head kidney was monitored by qPCR. To describe the immune reaction following this immunisation, a series of genes encoding cytokines, complement factors, immunoglobulins and acute phase reactants were studied. Genes encoding acute phase reactants in the liver were up-regulated with serum amyloid A (SAA) as the most pronounced with a 2299-fold increase at 24 h p.i. Hepcidin and pre-cerebellin were also up-regulated in the liver 24 h p.i., by 7- and 4-fold, respectively. Complement factors C3, C5 and factor B (Bf) were up-regulated in the spleen and the head kidney 24 h and 28 d p.i. Genes encoding immunoglobulins were not up-regulated, but a specific low titer IgM response (titer 25) against parasite antigens was detected by a modified ELISA 4 wk p.i.     

黄颡鱼自噬相关基因 LC3B和Beclin1的原核表达及多克隆抗体制备

为深入研究黄颡鱼Pelteobagrus fulvidraco自噬的发生过程和机制, 从黄颡鱼cDNA中克隆获得372和1344 bp的黄颡鱼LC3B(PfLC3B)和Beclin1(PfBeclin1)基因编码序列, 并成功构建了pET32a(+)-PfLC3B和pET32a(+)-PfBeclin1重组表达载体, 分别采用亲和层析和包涵体纯化的方法得到了纯度较高的重组PfLC3B和PfBeclin1蛋白; 以重组PfLC3B和PfBeclin1蛋白免疫Balb/C小鼠, 制备多克隆抗体。通过Western blot鉴定了PfLC3B和PfBeclin1抗血清可以分别特异性识别重组PfLC3B和PfBeclin1蛋白; PfLC3B抗血清可以特异性识别黄颡鱼内源性LC3-Ⅰ和LC3-Ⅱ蛋白, PfBeclin1抗血清可以特异性识别黄颡鱼内源性Beclin1蛋白。蛋白水平的组织分布显示, 黄颡鱼LC3和Beclin1蛋白在肝脏中表达水平均较高, 在肾脏和脾脏中的表达水平较低。综上所述, 研究成功制备了黄颡鱼LC3B和Beclin1的多克隆抗体, 为深入研究黄颡鱼自噬机制提供了有力工具。     

Confirmation of the chromosomal localization of human lamp genes and their exclusion as candidate genes for Salla disease

Summary Salla disease is an inherited lysosomal storage disorder caused by accumulation of free sialic acid in the lysosomes. Lamp genes, lamp A and lamp B (lysosome associated membrane proteins), are the first known genes encoding for human lysosomal membrane proteins. Absence of linkage in a large group of families shows that lamp genes are not involved in Salla disease. The lamp genes were localized, using Southern hybridization in hamster — human hybrid cell panels, to chromosomes 13 (lamp A) and X (lamp B).